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Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1005

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Pamoplantar Pustulosis.

Pathogenetic Studies with Special Referenceto the Role of Nicotine

BY

EVA HAGFORSEN

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001


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Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) inDermatology and Venereology presented at Uppsala University in 2001

ABSTRACTHagforsen, E. 2001.Palmoplantar pustulosis. Pathogenetic studies with special

reference to the role of nicotine. Acta Universitatis Upsaliensis.ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Medicine 1005. 56 pp.Uppsala. ISBN 91-554-4955-7.

Palmoplantar pustulosis (PPP) is a chronic disease of unknown pathogenesis. Most of the patients were smokers. High prevalence of a number of autoimmune diseases wasobserved among the patients (thyroid disease 14%, gluten intolerance 8%, diabetestype 1 3%).

Eosinophils and neutrophils were found in large numbers in the pustules. Massiveinfiltrates of lymphocytes and mast cells in the dermis below the pustule and anabnormal acrosyringial pattern indicate that the acrosyringium is the target for theinflammation. Immunofluorescence (IF) revealed decreased innervation of the sweatgland, outward migration of substance P-positive granulocytes in the acrosyringiumand an increased number of contacts between mast cells and nerve fibres in thedermis.

Distributions of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE)were studied, since they regulate the level of acetylcholine, the main inducer of sweating. The most intense AChE-like immunoreactivity (LI) was observed in theacrosyringium in the lowest part of the stratum corneum, corresponding to the site of the pustule in PPP. ChAT-LI in granulocytes and AChE-LI in mast cells weredemonstrated, which may have implications for inflammatory processes in general.

Nicotinic acetylcholine receptors (nAChR) are activated by acetylcholine but also by nicotine. Immunohistochemstry of -3 and-7 subtypes of the nAChRs showedthat the nAChR expression in healthy skin was influenced by smoking. A highlyabnormal -7 nAChR distribution in PPP skin was observed.

The levels of nAChR antibodies were elevated in 42% of the PPP sera, and 68% of these sera gave specific endothelial IF in the papillary dermis in skin from non-smokers. Positive IF in the acrosyringium was also noted in skin from smokers.

Conclusions:Smoking seems to induce up-regulation of an antigen in palmar skin.The results indicate that PPP is an autoimmune disease and that nicotine might have arole in the onset of the inflammation.

Key words:Palmoplantar pustulosis, smoking, sweat gland apparatus, neuropeptides,non-neuronal cholinergic system, nicotinic receptor antibodies, autoimmune disease.

Eva Hagforsen, Section of Dermatology and Venereology, Department of Medical Sciences, Uppsala University, University Hospital, SE-751 85 Uppsala, Sweden

Eva Hagforsen 2001

ISSN 0282-7476ISBN 91-554-4955-7

Printed in Sweden by Fyris-Tryck AB, Uppsala 2001


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and Johan, Martin, and Emma

Att vgar att frlora fotfstet fr en stund Att inte vgar att frlora sig sjlv

Sren Kierkegaard


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CONTENTS

ABBREVIATIONS.. 7PAPERS INCLUDED.. 8INTRODUCTION.. 9

Palmoplantar pustulosis . 9 Inflammation 10

Autoimmunity . 12Normal histology of palmar and plantar skin ... 12

The eccrine sweat gland apparatus 13 Normal histology and function 13

Inflammation and the acrosyringium. 14Innervation.. 14

Interaction between nervous and immune systems .. 15The neuronal cholinergic system 15The non-neuronal cholinergic system 17

General ... 17The non-neuronal cholinergic system in the skin 18Effects of acetylcholine on cells.. 19

Nicotinic influence- on the neuronal chol inergi c system . 20- on the non-neur onal chol in ergic system . 20- on keratinocytes 21- on endothel ial cel ls ... 21

AIMS OF THE STUDY. 22

PATIENTS AND METHODS... 23Patients. 23

Paper I, II, III and IV.. 23 Anamnestic data.. 23

Clinical examination 23Blood samples.. 23Biopsies 23

Paper V 23Reference persons 24Immunohistochemistry 24

Peroxidase and alkaline phosphatase methods.. 24Immunofluorescence... 26Serum immunofluorescence 27

Western blot (paper III) 28Radioimmunoassay (paper V). 28Statistics 29


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RESULTS AND DISCUSSION. 30Paper I ... 30

Anamnestic data. 30Clinical findings.. 32Inflammatory cells.. 32

The sweat gland and duct ... 33Paper II . 34 PGP 9.5, substance P and calcitonin gene-related peptide 34

Nerve fibres and their contacts with mast cells.. 35Neuropeptide immunoreactivity in granulocytes 36

Paper III 37 ChAT and AChE in the epidermis and sweat gland apparatus.. 37

ChAT and AChE in inflammatory cells... 39Paper IV 40

Nicotinic receptors in the epidermis and sweat gland apparatus.. 40Nicotinic receptors in inflammatory cells 42

Paper V.. 43 Serum antibodies to nicotinic receptors and the immunofluorescence pattern43

CONCLUSIONS. 45

ACKNOWLEDGEMENTS 46

REFERENCES 48


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ABBREVIATIONS

ab antibody

ABC avidin-biotin complex

ACh acetylcholine

AChE acetylcholinesterase

APAAP alkaline phosphatase-anti-alkaline phosphatase

C5a complement factor 5a

CGRP calcitonin gene-related peptide

ChAT choline acetyltransferase

ECP eosinophil cationic protein

EPO eosinophil peroxidase

EPX/EDN eosinophil protein X / eosinophil derived neurotoxin

FcRI high affinity receptor for immunoglobulin E

FITC fluorescein isothiocyanate

HLA human leukocyte antigen

IF immunofluorescence

Ig immunoglobulin

IL interleukin

LI like immunoreactivity

mAChR muscarinic acetylcholine receptor MC mast cells

MHC major histocompatibility complex

MNL mononuclear leucocyte

mRNA messenger ribonucleic acid

nAChR nicotinic acetylcholine receptor

PAP peroxidase anti-peroxidase

PBS phosphate buffered saline

PGP 9.5 protein gene product 9.5PPP palmoplantar pustulosis

SP substance P

TRITC tetramethyl-rhodamine-isothiocyanate

VIP vasoactive intestinal peptide


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PAPERS INCLUDED

I. Eriksson MO, Hagforsen E, Pihl-Lundin I, Michalsson G: Palmoplantar pustulosis-a clinical and immunohistochemical study. Br J Dermatol. 1998;138: 390-398.

II. Hagforsen E, Nordlind K, Michalsson G: Skin nerve fibres and their contactswith mast cells in patients with palmoplantar pustulosis. Arch Dermatol Res.2000; 292: 269-74.

III. Hagforsen E, Aronsson F, Einarsson A, Nordlind K, Michalsson G: Thedistribution of choline acetyltransferase- and acetylcholinesterase-likeimmunoreactivity in palmar skin from patients with palmoplantar pustulosis.Br J Dermatol. 2000; 142: 234-42.

IV. Hagforsen E, Edvinsson M, Nordlind K, Michalsson G: Expression of -3and -7 subunits of nicotinic acetylcholine receptors in the skin of patientswith palmoplantar pustulosis. (submitted)

V. Hagforsen E, Mustafa A, Lefvert AK, Nordlind K, Michalsson G:Antibodies against nicotinic receptors in serum from patients with palmoplantar pustulosis. (submitted)

Reprints were made with permission from the publishers.

Cover photograph: Immunofluorescence pattern with PPP sera on normal palmar skin from a non- smoking control. Note the pattern in the papillary dermis. A photograph of a palm from a PPP patient is inserted.


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INTRODUCTIONPalmoplantar pustulosisPalmoplantar pustulosis (PPP) is a chronic skin disease with an unknown

pathogenesis. It may be a localised form of pustular psoriasis, and occasionally the

patients have psoriasis-like lesions, particularly on the forearms and legs, but the

relationship is controversial. PPP is characterised by sterile intra-epidermal pustules

and usually also erythematous, scaly skin on the palms and soles. It is more common

in women than in men and is also more common in smokers than in non-smokers

(Eriksson et al 1998). The age at onset is usually between 20 and 60 years, 40-60

being most common.

Associations between PPP and autoimmune diseases such as autoimmune thyroid

disease have been reported (Rosn 1988). However, in that study no improvement of the palms and soles occurred when the thyroid disease was treated. A diabetic pattern

has been found in 22% of Japanese PPP patients at oral glucose tests (Uehara 1983)

but the clinical relevance of these tests is difficult to evaluate, since abnormal glucose

tolerance tests are not uncommon in middle-aged and elderly persons. Furthermore,

symptoms resembling rheumatoid arthritis have been noted in 13% of PPP patients,

which may be compared with a figure of 2.7% in the general population (Enfors and

Molin 1971).It is known that the pustules in PPP contain neutrophil granulocytes, but why these

cells are so abundant here is still unknown. There is a report, however, of intercellular

expression of interleukin-8 (IL-8) in the epidermis (Anttila et al 1992) of PPP skin

and IL-8 is a chemoattractant for neutrophils (Baggiolini et al 1989). Anttila et al also

observed strong IL-8 immunoreactivity in the whole eccrine sweat gland apparatus in

palmar skin from both PPP patients and control subjects. IL-8 has also been found in

sweat, and both this protein and its mRNA have been detected in sweat glandepithelium in abdominal skin (Jones et al 1995), indicating that IL-8 is produced in

situ.

There is no curative treatment for PPP today. Usually the patients with mild

symptoms are treated with emollients. Topical steroids are used in patients with more

severe forms and the most severe cases are treated with systemic drugs (retinoids,

cyclosporin). All these drugs are potent anti-inflammatory agents, which are virtually

independent of the cause of inflammation.


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Inflammation

There are two fundamentally different types of defence against infection and tissue

damage, namely the innate response and the adaptive response.

Neutrophils are called the first line of defence, since within minutes of tissue

damage or pathological invasion they adhere to the endothelium of vessel walls and

migrate into the involved tissue. These cells form part of the innate immune response.

Other cells involved in the innate response are monocytes and macrophages

(phagocytic cells), basophils, mast cells and eosinophils (which release inflammatory

mediators), and natural killer cells. Neutrophils and macrophages have receptors for

antibodies and complement, a fact which enhances phagocytosis of microorganisms

coated with immunoglobulin and/or complement. Phagocytes also remove the bodys

own dead or dying cells. The most important chemotactic factors for neutrophils are

C5a (derived from complement), bacterial products (such as N-formyl-methionyl-

leucyl-phenylalanine), leucotriene B4 (product of arachidonic acid metabolism) and

IL-8.

Eosinophils are mainly tissue cells, and are most abundant in the gastrointestinal

tract, skin and lungs. Eosinophils are involved in processes in, for example, allergy

and parasitic infections. They contain different granular structures, of which specific

granules are most numerous. The specific granules, in turn, contain eosinophil

cationic protein (ECP), eosinophil protein X (EPX) (also known as eosinophil derived

neurotoxin (EDN)) and eosinophil peroxidase (EPO). Activated eosinophils probably

kill parasites mainly by releasing ECP, instead of by phagocytosis. Eosinophils may

also induce neurotoxic effects by secreting EPX, and EPO has antibacterial

properties. In addition, eosinophils secrete prostaglandins, leukotrienes and various

cytokines. Lipids, complement components, cytokines and chemokines are known

eosinophil chemoattractants. Eotaxin is an example of a more recently discoveredeosinophil chemoattractant (Garcia-Zepeda et al 1996). Increased amounts of ECP in

the serum or in tissues have been shown to reflect increased turnover and/or increased

tissue activity of the eosinophils.

Basophils and mast cells (MC) are important in atopic allergies. Allergen binding to

IgE bound to high-affinity IgE receptors (FcRI) cross-links the FcRI, leading to

secretion of inflammatory mediators such as histamine, prostaglandins and

leukotrienes. Mast cells migrate into tissues, where they mature. They seem to be


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localised in organs that are potential ports of entry of foreign agents, such as the skin,

lungs and gut. Mast cells have been found in tissues of patients with allergic diseases,

but these cells are also linked to chronic inflammatory disorders with a hitherto

unknown pathogenesis, for example psoriasis. They are divided into subsets on the

basis of their content of neutral serine proteases (Irani et al 1986). One subset, MCTC,

contains tryptase, chymase, cathepsin G and carboxypeptidase, whereas the other

subset, MCT, contains only tryptase. MCTC is found predominantly in the skin and in

the bronchial, nasal and intestinal mucosa, whereas MCT is localised mainly in

mucosal surfaces (Irani et al 1986; Irani et al 1989).

B and T lymphocytes (helper and cytotoxic) are involved in the adaptive (cell-

mediated) response. When T cells develop (in the thymus), there is a positive

selection in which cells that are able to interact usefully with peptides presented by

major histocompatibility complex (MHC) molecules survive, and a negative selection

in which cells reactive to self-proteins undergo apoptosis. Only 1% of the immature

precursor cells develop to immunocompetent T cells and are passed into the

circulation. The antigen-presenting cell is another important cell, which displays the

antigen to the T-cell receptor on the surface of helper T lymphocytes. The antigen is

presented by MHC class II molecules on the surface of dendritic cells. There are three

main kinds of class II molecules, HLA-DR, -DP and -DQ. When the receptors of the

T cells bind to antigens, the antigen-specific T cells proliferate (clonal selection).

Cytotoxic T cells have an antigen-specific T cell receptor, which recognises antigens

bound to MHC class I molecules (HLA-A, -B or -C), which are expressed on virtually

all cells. Self-cells that have been altered or infected are recognised and destroyed by

cytotoxic T cells. Mature B cells (plasma cells) produce antibodies against specific

antigens presented to them by helper T cells. Five different classes of antibodies

(immunoglobulins) are produced. One of them, immunoglobulin A (IgA), has animportant role in the first defence of the mucosal surfaces.

Cytokines, small soluble proteins, play a central part in the communication between

cells in the immune system and are also important for growth and differentiation of

haematopoietic, epithelial and mesenchymal cells. They regulate cell function in pico-

to nano-molar concentrations through specific receptors. There are pro-inflammatory

cytokines, e.g. IL-1, tumour necrosis factor , IL-6 and granulocyte-macrophage

colony stimulating factor, inflammatory cytokines, e.g. IL-2, IL-4 and interferon-


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gamma (IFN ) and cytokines that are anti-inflammatory, e.g. IL-10, transforming

growth factor and IL-1 receptor antagonist. All these cytokines are involved to

varying degrees in different types of skin inflammation, including autoimmune

reactions, and are thus believed to have a role in the pathogenesis of psoriasis andrelated skin diseases.

AutoimmunityA disease is defined as autoimmune if the tissue damage is shown to be caused by an

immune response to self-antigens. Autoimmune disease can be triggered by

autoreactive T cells or by autoantibodies. The tissue may be damaged as a result of a

direct attack on the cells bearing the antigen, of immune complex formation or of

local inflammation. Another type of autoimmunity occurs when autoantibodies bind

directly to cellular receptors, causing either excess activity (e.g. Graves disease) or

inhibition of receptor function (e.g. myasthenia gravis).

Autoimmunity can be triggered by a variety of mechanisms, in most of which

infectious agents are involved. As a result of a tissue injury, antigens that are not

normally present in the circulation may be exposed to the immune response and not

recognised as a self-antigen. Infectious agents may induce either T- or B-cell

responses that can cross-react with self-antigens (molecular mimicry). Superantigens

produced by bacteria or viruses bind directly to the MHC class II molecule and

induce polyclonal T-cell activation leading to an autoimmune process.

Many human autoimmune diseases show HLA-linked associations, as may be

expected, since the ability of T cells to respond to a particular antigen depends on the

MHC type. It also seems as if sex hormones are involved in the pathogenesis of

autoimmune diseases. For example, systemic lupus erythematosus is more common in

women.

Normal histology of palmar and plantar skinIt is not known why PPP is localised to the palms and soles. However, the skin in

these locations differs from that in other parts of the body. Glabrous (non-hairy) skin

is characterised by a thick epidermis divided into several well-marked layers. There

are no hair follicles in glabrous skin, nor are there any sebaceous glands. In the

dermis of glabrous skin there are encapsulated sense organs, whereas the sensory


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nerve endings in hairy skin are sometimes free, or terminate in hair follicles, and

others have expanded tips. The density of the sweat glands on the palms and soles is

600 to 700/cm2, compared to only 64 glands/mm2 on the back. Furthermore, the

stratum corneum is much thicker on the palms and soles, as a result of which the

outermost part of the sweat duct (the acrosyringium) has a well-developed coil

structure there, which is not so apparent in other sites.

The eccrine sweat gland apparatus Normal histology and function

The eccrine sweat gland apparatus consists of a secretory coil and a duct (Fig.1). The

coiled part is made up of the secretory coil and the proximal duct. The distal duct is

straight and connects the coil with the epidermis. In the epidermis and the stratum

corneum the duct forms a spiral (the acrosyringium) leading up onto the skin surface.

There are two main types of sweating: thermoregulatory and emotional (mental)

sweating. Thermoregulatory sweating occurs especially on the upper part of the trunk

and the face, but also on the palms and soles. Emotional sweating is provoked by

anxiety or pain and is characteristically associated with the palms, but its underlying

mechanisms are not known. The nerves surrounding the sweat glands are sympathetic

post-ganglionic fibres, which consist of non-myelinated class C nerve fibres, but


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acetylcholine (ACh) is the principal neurotransmitter, acting via muscarinic receptors.

However, adrenaline may also induce palmar eccrine sweating (Wolf and Maibach

1974). Nicotine has been found capable of inducing axon reflex sweating by

iontophoresis (on the foot and leg) and produced a stained area (iodine starch

method) similar to that after ACh iontophoresis (Riedl et al 1998).

Inflammation and the acrosyringium

The acrosyringial epithelium possesses specialised keratinocytes, which are immune-

competent. Accordingly, expression of MHC class II HLA-DR, which play a critical

role in cell-mediated immune responses, has been observed on acrosyringial

epithelium in normal human skin (Murphy et al 1983). In a study of the antigenic

profile of the acrosyringium in normal skin (from the abdomen), McGregor el al

(1991) found expression of HLA-DR, -DP and -DQ on the acrosyringial epithelium,

and on the keratinocytes surrounding the acrosyringium they observed CD 36

(monocyte/platelet-specific molecule). Furthermore, expression of CD 68

(monocyte/macrophage-specific molecule) was detected on the acrosyringial

epithelium, but not on dermal ducts or sweat glands. Immunohistochemically,

Reitamo et al (1990) demonstrated IL-1 throughout the eccrine sweat gland apparatus.

The distal part of the acrosyringial epithelium showed intense staining. Didierjean et

al (1990) reported the presence of IL-1 in sweat from both truncal and palmar-

plantar regions, whereas IL-1 was detectable only in sweat from palms and soles,

indicating a site-dependent difference in the secretion of the two IL-1 molecules.

Furthermore, the IL-1 concentrations were much higher in the sweat during jogging

and sauna bathing than during spontaneous sweating, which they suggested could be

due to a stress-induced increase in the production of IL-1 by sweat gland cells. IgA,

which forms a defence barrier against microbial antigens on mucosal surfaces, has been detected in sweat secreted onto the skin surface (Imayama et al 1995), indicating

involvement of this immunoglobulin in the local immune defence of the skin.

Innervation

Most nerve fibres in the skin are sensory, and most of them are unmyelinated C and

myelinated A fibres that end as free nerve endings. Sensory nerve fibres express

several neuropeptides, which are biologically active polypeptides, and most


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neuropeptide-containing fibres are located around blood vessels, sweat glands and

hair follicles or are present as free nerve endings. Neuropeptides can induce

neurogenic inflammation. Substance P (SP), calcitonin gene-related peptide (CGRP)

and vasoactive intestinal polypeptide (VIP), among other peptides, have been

demonstrated in nerve fibres in human skin (Wallengren et al 1987). Substance P-

containing fibres are most densly located in the palms, soles and axillary skin (Eedy

1993).

Human sweat glands are entangled with nerve fibres. Along the sweat duct, from

the gland to the surface of the skin, one or two nerve fibres are oriented. The nerve

fibres

around the sweat gland apparatus are reported to express CGRP, VIP and sparsely SP

(Kennedy et al 1994).

Interaction between nervous and immune systemsThere have been many reports indicating that the nervous system interacts with the

cutaneous immune system to mediate local inflammation. For instance neuropeptides

might be involved in skin diseases such as psoriasis [CGRP (Artemi et al 1997), SP

(Al'Abadie et al 1995); (Naukkarinen et al 1996), VIP (Anand et al 1991)], atopic

dermatitis [CGRP (Pincelli et al 1990), VIP (Ostlere et al 1995; Pincelli et al 1991)]

and eczema [VIP (Anand et al 1991)].

Some neuropeptides are able to degranulate mast cells (for example SP and VIP;

(Ebertz et al 1987; Lowman et al 1988) and also to induce vasodilatation and may

stimulate chemotactic and phagocytic activity of neutrophils and stimulate IgA

production by B cells [reviewed in Ansel et al (1996)].

It has been shown that neutrophil granulocytes contain VIP (ODorisio et al 1980),

that mast cells in patients with atopic dermatitis contain SP (Toyoda et al 2000) andthat activated T cells also contain SP (De Giorgio et al 1998).

The neuronal cholinergic system

Acetylcholine is the neurotransmitter of the cholinergic system. The synthesis of ACh

from coenzyme A and choline is catalysed by choline acetyltransferase (ChAT).

Acetylcholinesterase (AChE) is the cholinergic enzyme that hydrolyses ACh to

acetate and choline.


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Acetylcholine acts on cells via two different classes of receptors, nicotinic (nAChR)

and muscarinic acetylcholine receptors (mAChR). Receptors of both classes are

found in the central nervous system.

Nicotinic receptors are also present in autonomic ganglia and at neuromuscular

junctions, while muscarinic receptors are found on autonomic effector cells

innervated by post-ganglionic parasympathetic nerves, and in blood vessels, where

they modulate vasoconstriction and dilatation (Furchgott and Zawadzki 1980).

The nicotinic AChRs are ligand-gated ion channels that mediate influx of Na+ and

Ca2+ and efflux of K + and are formed by various combinations of transmembrane

, , , and glycoprotein subunits. Each nAChR consists of five such subunits,

different combinations of which determine the functional and pharmacological

characteristics of the receptor (Conti-Tronconi et al 1994). The 1, 1, , and

subunits have been found at the neuromuscular junction. Neuronal nAChR consists of

combinations of the -2 to -9 and -2 to -5 subunits. Nicotinic AChRs formed by the

-4 and-2 subunits are the major subtype in the brain (Whiting et al 1991). The-3

subunit generally forms nAChRs together with the-5, -2, and-4 subunits (Conti-

Tronconi et al 1994). The-7, -8 and -9 subunits can form functional nicotinic

receptor channels of their own. The ACh-binding sites are believed to reside

primarily on the subunits (Papke 1993). However, -5 subunits, which are closely

related to-3 subunits, are believed not to be capable of forming an ACh-binding site

(Wang et al 1996). Alpha-7 nAChRs, which have five subunits, thus have five

putative binding sites for ACh (Fig. 2). It has also been demonstrated that -7

nAChRs are highly permeable to Ca2+ (Bertrand et al 1993).

Fig. 2. Subunit arrangements of two nAChR types around the central cation channel. To the left, thesubtype with3 as binding site with two putative ACh-binding sites; to the right, subtypes (e.g.7,8 or 9) forming functional homomers with five putative ACh-binding sites.


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The muscarinic AChRs are glycoproteins with seven-helical transmembrane

segments, and they are coupled to G proteins (Hulme 1990; Hulme et al 1990). There

are five known subtypes, m1 to m5. The muscarinic receptor subtypes m1, m3 and

m5 mediate, upon stimulation, an activation of phospholipase C activity, resulting inan increase in the Ca2+ concentration; m2 and m4 mediate, upon stimulation,

inhibition of adenyl cyclase activity, resulting in decreased cyclic AMP formation

(Hulme 1990; Hulme et al 1990).

Neuronal nAChRs may be involved in neuronal diseases. It has been suggested, for

example, that-7 nAChRs may be associated with some aspects of schizophrenia.

Freedman et al (1995) found a decrease in -7 nAChR in the hippocampus of brains

from schizophrenia patients, and a decreased level of the -7 nAChR subunit proteinhas also been observed in the frontal cortex of schizophrenic brain (Guan et al 1999).

A high proportion of schizophrenic patients are intensive tobacco users (Lohr and

Flynn 1992), and it has been proposed that they may be attempting to self-medicate

(Dalack et al 1998). In patients with Alzheimers disease a decrease in high-affinity

nicotine binding sites is one among other changes in the brain (Nordberg and

Winblad 1986; Whitehouse et al 1986). Parkinsons disease is also associated with a

large loss of high-affinity nicotine binding sites in the brain (Perry et al 1995).

The non-neuronal cholinergic systemGeneral

The term non-neuronal cholinergic system is based on the fact that ACh is found in

cells other than neurones. Dale (1914) and Ewins (1914) obtained the first evidence

of the presence of ACh in plants.

The occurrence of acetylcholine has been analysed in human placenta (Rowell and

Sastry 1981), bronchial epithelial cells (Klapproth et al 1997; Wessler et al 1995)

mononuclear cells (Fujii et al 1996), sperm (Sastry and Sadavongvivad 1978), retina

(Hutchins and Hollyfield 1986) and keratinocytes (Grando et al 1993b; Klapproth et

al 1997).

A variety of non-neuronal tissues synthesise and degrade ACh. Expression of the

ChAT protein has been found in non-neuronal cells such as bronchial epithelial cells

(Klapproth et al 1997; Reinheimer et al 1996), keratinocytes (Grando et al 1993b),


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cells of the human small and large intestine (Klapproth et al 1997) and placental cells

(Rowell and Sastry 1981).

AChE has also been found in the placenta (Rowell and Sastry 1981) and in

heamatopoietic cells, i.e. red blood cells (Herz and Kaplan 1973), platelets and T

lymphocytes, but not B lymphocytes (Szelenyi et al 1982). In a colonic biopsy

specimen, expression of AChE mRNA has been detected in mast cells with high-

affinity receptors for IgE (Nechushtan et al 1996).

Furthermore, some of these cells have been shown to express ACh receptors.

Macklin et al (1998) have reported that human vascular endothelial cells express the

subunits that form functional nAChR similar to the nAChR expressed by ganglionic

neurones ( -3, -5, -2 and-4). These subunits have also been detected in human

bronchial epithelial cells (Maus et al 1998), where Zia et al (1997) found the-3, -

4, -5 and -7 subunits. The alpha-7 subunit of nAChR is also expressed by

endothelial cells (Conti-Fine et al 2000), and the -3 and -4 subunits have been

found on lymphocytes (Hiemke et al 1996).

The muscarinic receptor subtypes m2 and m3 have been found in human

mononuclear leucocytes (MNLs) (Bronzetti et al 1996). Hellstrm-Lindahl and

Nordberg (1996) found the mRNAs for the m3, m4 and m5 muscarinic subtypes in

MNLs and also in purified T cells. Fujino et al (1997) observed expression of the m1

and m2 subtypes in human lymphocytes. Human skin fibroblasts have been reported

to express m2, m4 and m5 mAChR subtypes (Buchli et al 1999).

The non-neuronal cholinergic system in the skin

Grando et al have made detailed investigations of the non-neuronal cholinergic

system in the skin. They demonstrated that human keratinocytes express the ChAT

protein and also synthesise and secrete ACh (Grando et al 1993b). Furthermore, they

showed that the human epidermis expresses AChE and also nicotinic and muscarinic

AChR (Grando 1997; Grando et al 1995a). The ion channels on keratinocytes are

similar to those expressed by ganglionic neurons, since the-3, -5, -2 and-4

subunits, which are found on keratinocytes, are known to form functional receptors in

several combinations among themselves (e.g.32, 325, 34, 345 or

3245) on ganglionic neurons, and the-7 subunit, which is also present on

keratinocytes, can form functional nAChRs of its own (Grando et al 1995a).


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Keratinocyte ACh, like neuronal ACh, uses Ca2+ as a second messenger (Grando et al

1996). Ion fluxes mediated by nAChR channels are essential for maintaining

keratinocyte viability, as demonstrated in experiments using muscarinic and nicotinic

blocking agents, where interruption of nicotinic, but not muscarinic, pathways of ACh

signalling was found to inhibit keratinocyte division and result in premature cell death

(Grando et al 1993a). Both nicotinic and muscarinic AChRs regulate cell adhesion

and motility, since blocking by -bungarotoxin and mecamylamine, specific AChR

antagonists, caused cell detachment and abolished cell migration (Grando et al

1995a).

High affinity mAChRs have been found on keratinocyte cell surfaces in a high

density (Grando et al 1995b). These receptors mediate effects of muscarinic drugs on

keratinocyte viability, proliferation, adhesion, lateral migration and differentiation.

The mAChR subtypes m1, m3, m4 and m5 have been found in the epidermis (Ndoye

et al 1998). Keratinocytes expressed a unique combination of mAChR subtypes at

each step of their development in the epidermis and it was proposed that each

receptor may regulate a specific cell function (Ndoye et al 1998).

Effects of acetylcholine on cells

Non-neuronal ACh can exert its effects through different pathways. Acetylcholine

released from non-neuronal cells activates the membrane-bound AChR (nicotinic and

muscarinic) localised on the same (autocrine effects) or on neighbouring cells

(paracrine effects). Klapproth et al (1997) reported that the mitogenic effect of ACh

on cultured human bronchial epithelial cells was counteracted by antagonists of

nicotinic and muscarinic receptors. Thus, the effect of ACh is mediated by the

classical extracellular membrane-bound receptors. Interestingly, in the same study it

was found that the ChAT blocker, bromoacetylcholine, had a stronger antiproliferative effect than a combination of the two antagonists blocking the

nicotinic and muscarinic receptors respectively, which may suggest that ACh also has

cytosolic action.

Experimental evidence has been presented that ACh is involved in the regulation of

the mitotic cycle of epithelial cells in humans (Cavanagh and Colley 1989; Grando et

al 1995a; Grando et al 1993b; Klapproth et al 1997). Furthermore, detection of ACh

and its receptors in immune cells indicates that it may take part in the immune system


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20

by activation and proliferation of these cells [reviewed in Kawashima and Fujii

(2000)].

Nicotinic influence on the neuronal chol inergi c system

Nicotine acts agonistically on nAChR, and is thus able to reproduce the same effects

as ACh on cells expressing these receptors, but in contrast to ACh, nicotine is not

degraded by AChE. The nAChR channel opens in response to the binding of agonist

(activation) but also becomes refractory to activation during prolonged exposure to

nicotinic agonists (desensitisation) (Peng et al 1994). The nicotine concentrations

required to desensitise the receptor are nearly 1000 times lower than those required

for activation [reviewed by Changeux (1990)]. It has been reported that smoking

increases the number of nAChRs in the human brain (Breese et al 1997). It is

proposed that nicotine-induced up-regulation of neuronal nicotinic receptors results

from a decrease in the rate of receptor turnover (Peng et al 1994). The increase may

also be due to an adaptive response of neurones to accumulation of chronically

desensitised receptors. Alpha-3 nAChRs are more resistant to desensitisation than

other receptor subtypes, such as those containing the -4 and -7 subunits (Olale et al

1997).

on the non-n eur onal choli nergic system

Nicotine is present in high concentrations in the blood of smokers (Russell et al 1980)

and might contribute to desensitisation of the nAChRs and in this way influence their

normal function. There are reports that smoking increases the number of nAChRs on

human bronchial epithelial cellsin vivoand that nicotine increases the number of

nAChRsin vitroon these cells (Zia et al 1997), and that long-term exposure of

respiratory epithelial cells to nicotine increases their Ca2+ concentration, which could

lead to cell damage. Smoking also increased the number of nAChRs on

polymorphonuclear cellsin vivo(Benhammou et al 2000; Lebargy et al 1996)

Lebargy et al (1996) reported that the increase in binding sites persisted for several

months and did not return to the non-smoking level until one year after cessation of

smoking.


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21

Alpha-7 nAChRs have been found in small cell lung carcinomas, and the growth of

cell lines derived from these tumours was inhibited by an -7-specific antagonist

(Chini et al 1992; Codignola et al 1996; Quik et al 1994). Thus, in small cell lung

carcinomas in smokers the cancer growth may be facilitated by nicotine stimulationof -7 receptors.

on keratinocytes

Short-term exposure to nicotine stimulates cytoplasm motility and lateral migration of

cultured keratinocytes (Grando et al 1995a). Other (keratinocyte) functions such as

proliferation, adhesion and differentiation may also be affected as a result of

accelerated ion exchange through nicotinic channels. Chronic exposure to nicotineabolishes migration of cultured human keratinocytes in a dose-dependent manner

(Lee et al 1996). Furthermore, chronic nicotine exposure leads to an increase in the

number of keratinocytes forming cornifying envelopes, as well as in the expression of

filaggrin, involucrin and transglutaminase type 1 (Grando et al 1996). Zia el al (2000)

found that keratinocytes incubated with nicotinein vitroexpressed a higher

percentage of the -7 nAChR.

De Hertog et al (2001) concluded that tobacco smoking is probably a risk factor for

cutaneous squamous cell carcinoma. Current smokers were found to be at higher risk

than former smokers, and a clear relation to the number of cigarettes currently

smoked was also observed.

on endotheli al cell s

Nicotine is known to induce vasoconstriction. The cutaneous blood flow, as measured

with a laser Doppler flowmeter, was decreased both in habitual smokers and in non-

smokers after smoking a single cigarette (Monfrecola et al 1998). The micro-

circulation showed a slower recovery phase in the smokers, however, indicating

adaptation to smoke.

There are functional nicotinic receptors on the endothelial cells (Macklin et al

1998). Since in tobacco users the concentration of nicotine in the blood is high

(Russell et al 1980), these receptors may become desensitised after prolonged

exposure to nicotine, which can make them unable to respond in a normal way to the

endogenous ACh.


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22

AIMS OF THE INVESTIGATION

The general aim of this investigation was to study the pathogenesis of palmoplantar

pustulosis.

The specific aims were:

- to define the cellular components of the inflammation in PPP.

- to localise the site of inflammation with particular reference to the eccrine gland

and duct.

- to study the distribution of the general nerve marker PGP 9.5 and of the

neuropeptides substance P and calcitonin gene-related peptide in PPP skin.

- to study contacts between sensory nerve fibres and mast cells in PPP skin.

- to study the distributions of choline acetyltransferase and acetylcholinesterase in

palmar skin from healthy non-smokers and smokers and from PPP patients.

- to study the distributions of the -3 and -7 subunits of the nicotinic

acetylcholine receptor in palmar skin from healthy non-smokers and smokers, and

from PPP patients.

- in view of the association between PPP and autoimmune diseases, to address the

question of whether PPP itself might be an autoimmune disease by measuring

serum antibodies to nAChR and by screening PPP sera for immunofluorescence

in healthy palmar skin.


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23

PATIENTS AND METHODS

PatientsPaper I, II, III and IV

Anamnestic data

Fifty-nine patients (52 women, 21-79 years old, and 7 men, 43-71 years old) with

typical PPP of the palms and/or soles answered a questionnaire. Their smoking habits

over the years was also investigated.

Clinical examination

Thirty-nine of the patients (35 women, 4 men) who answered the questionnaire were

examined clinically (GM). The degree of erythema and scaling was graded from 0 to

4 and the number of fresh pustules was counted. The patients were only using

emollients at the time of the examination. None of the patients were taking beta-

blockers or lithium.

Blood samples

Sera from the patients were analysed by routine methods for: triiodothyronine,

thyroxine, thyroid stimulating hormone, immunoglobulins (IgG, IgA, IgM and IgE),

eosinophilic cationic protein and antibodies (ab) to thyroglobulin, thyroid peroxidase,

parietal cells and gliadin (IgA and IgG).

BiopsiesAfter intradermal injection of xylocaine-adrenaline, one to three 3-mm punch biopsy

specimens were taken from involved skin and in some patients also one from

seemingly non-involved skin.

The specimens were either fixed in buffered 4% formalin and embedded in paraffin,

or snap-frozen in 70oC, or fixed in 4% paraformaldehyde with 0.2% picric acid

(Lanas fixative) for one hour and then rinsed in 0.1 M Srensens buffer containing

10% sucrose for at least 24 h before they were frozen.

Paper V

In this study sera from the 39 patients described above and six new sera were used

(39 women, 19-71 years old; 6 men, 36-70 years old). At the onset of PPP 43 patients

were smokers. At the time of the present study 9 had stopped smoking or had reduced

the number of cigarettes in recent years.


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24

Reference persons

The reference group consisted of two subgroups: smokers and non-smokers. All of

the smokers had been smoking for many years.

The number of smokers and non-smokers varied in the different studies:

Papers I and II: 2 smokers (1 woman, 1 man) and 7 non-smokers (6 women, 1 man),

all of them healthy.

Papers III and IV:7 smokers (5 women, 2 men) and 8 non-smokers (7 women, 1

man), all of them healthy.

Three punch biopsy specimens, which were handled in the same way as the

specimens from the patients, were taken from palmar skin in all of these persons.

Paper V: In this study serum samples were taken from 23 patients with palmar

eczema. Of these patients, 15 had smoked for many years, but six of them had

stopped smoking in recent years.

One 3-mm skin punch biopsy specimen was taken from healthy non-smoking and

smoking persons, from the hypothenar region after intradermal injection of xylocaine-

adrenaline. For comparison, biopsy specimens were also taken from the dorsal aspect

of the forearm and from the gluteal region. These specimens were snap-frozen at

-70oC.

Immunohistochemistry Peroxidase and alkaline phosphatase methods

Detailed descriptions of the different methods used in these studies are given in the

respective papers.

Table 1a presents an overview of the different antibodies used, the fixatives, and thestaining techniques employed.

In all specimens endogenous peroxidase activity was blocked by incubation in 0.3%

H2O2 in phosphate buffered saline (PBS) for 15 min. Between the incubations the

sections were rinsed in PBS twice for 5 min.

Controls with IgG of the same isotype and in the same dilution as the primary

monoclonal antibodies were negative. Polyclonal antibodies gave no staining when

they were preabsorbed with their corresponding peptides.


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25

T a b

l e 1 a

. A n t

i b o d

i e s u s e d

i n p e r o x i

d a s e a n

d a l

k a l i n e p h o s p h a t a s e m e t

h o d s

.

P a p e r

n u m

b e r

A n

t i g e n

A n t i b o d y

V i s u a l

i z i n g

D i l u t i o n

S o u r c e

F

i x a t

i v e

T e c

h n

i q u e

I

E o s

i n o p

h i l c a t

i o n i c p r o t e i n

A n t i - E G 2

E o s

i n o p

h i l s

1 / 2 0 0

K a b

i P h a r m a c

i a

A

c e t o n e

P A P

A P A A P *

I

H u m a n n e u t r o p h

i l l i p o c a l

i n

( H N L ) ( S e v e u s e t a l

1 9 9 7 )

A n t i - H N L

N e u

t r o p

h i l s

1 0 u g / m

L

P h a r m a c

i a D i a g n o s

t i c s

M

e t h a n o

l

A P A A P *

I

C D 3

A n t i - C D 3

L y m p h o c y t e s

1 / 1 0 0

B e c

t o n -

D i c k i n s s o n

A

c e t o n e

P A P

I

T r y p

t a s e

M A B 1 2 2 2

M a s

t c e l

l s

1 / 5 , 0 0 0

C h e m

i c o n

I n t . I n c .

F o r m a l

i n

P A P

I

K e r a t

i n s

( W a t a n a b e e t a l

1 9 9 3 )

A E 1 / A E 3

S w e a

t g l a n d

a p p a r a

t u s

1 / 1 , 0 0 0

B o e

h r i n g e r M a n n h e i m

C o r p .

F o r m a l

i n

P A P

I I I

C h A T

A n t i - C h A T

C h A T

1 / 5

B o e

h r i n g e r M a n n h e i m

C o r p .

L a n a

A B C * *

I I I

C h A T

M A B 3 0 5

C h A T

1 / 2 5 0

C h e m

i c o n

I n t . I n c .

A

c e t o n e

A B C

I I I

A C h E

M A B 3 0 3

A C h E

1 / 6 0 0

C h e m

i c o n

I n t . I n c .

A

c e t o n e

A B C

I V

- 3 n A

C h R s u

b u n i

t

m A B 3 1 3

n A C h R

1 / 3 , 0 0 0

R B I

L a n a

A B C * *

I V

- 7 n A

C h R s u

b u n i

t

A C h R - 7

n A C h R

1 / 5 0

S a n t a

C r u z

I n c .

A

c e t o n e

A B C

* T h e a l k a

l i n e p h o s p h a t a s e a n t

i a l k a l

i n e p h o s p h a t a s e

( A P A A P ) t e c h n i q u e w a s u s e d , a

s n e u t r o p

h i l s c o n t a i n p e r o x i

d a s e , w

h i c h c o u l

d g i v e

f a l s e p o s i

t i v e s t a i n i n g .

* * S e c

t i o n s u s e d w e r e

1 4 u m , i

n o t

h e r s

t a i n i n g s

6 u m s e c t

i o n s w e r e u s e d .

T h e n u m

b e r o

f s t a i n e d c e

l l s w a s c o u n

t e d i n t h e e p

i d e r m

i s , p

a p i l l a r y

d e r m

i s ( b e l o w

t h e p u s t u l e w

h e n a p p l

i c a b

l e ) a n d

i n t h e r e

t i c u l a r d e r m

i s .

T h e s e c t

i o n s s t a i n e

d

w

i t h A E 1 / A E 3 w e r e

t r e a

t e d w

i t h 0 . 0 5 % p r o t e a s e f o r

1 0 m

i n b e f o r e s t a i n i n g . A

E 1 / A E 3 c o n

t a i n s a n

t i b o d

i e s a g a i n s

t c y t o k e r a t

i n s

1 - 8 , 1 0

, 1 4 / 1 5

, 1 6 , a n

d 1 9

. S e c

t i o n s

f r o m

t y l o t i c e c z e m a w e r e a l s o s t a i n e

d w

i t h A E 1 / A E 3 f o r c o m p a r i s o n .

T h e A

C h R - 7 a n

t i b o d y w a s

t h e o n

l y o n e

t h a t w a s p o

l y c l o n a l .


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26

To reduce the possible variations in staining intensities, all specimens used for one

antibody were stained on the same day. All immunohistochemical and

immunofluorescence evaluations were made on coded slides.

In the vital epidermis the staining intensity was estimated in the different strata as

follows: unstained = 0, weak = 1, medium = 2 and strong = 3. The numbers of

unstained and of ChAT-, AChE-,-3- and -7-positive ducts in the reticular dermis,

in the papillary dermis and in the vital epidermis and coils were counted. All visible

dermal ducts were counted as one duct each. The proportions of weakly and strongly

stained coils and ducts in the reticular dermis were calculated by dividing their

number by the total number (unstained and stained) of coils and ducts in the reticular

dermis. The numbers of immunoreactive cells in the papillary dermis and reticular

dermis and below the pustules were counted and classified as very few (0-3), few (4-

10) or many (>10).

One immunohistochemical double staining was performed (Table 1b).

Table 1b. Double staining with ABC and APAAP techniques.

Paper number Antigens Visualising Techniques

III AChE and chymase1 AChE in mast cells ABC APAAP

1The chymase antibody was used to verify that only mast cells were AChE+, since this antibodyworked better than the tryptase antibody in this double staining. Both antibodies were monoclonal.Sections 6m thick were used.

Immunofluorescence

Table 2a shows the different antibodies used, the fixatives, and the staining

techniques employed. Five non-adjacent sections were placed on each slide.

Table 2a. Antibodies used in immunofluorescence stainings.

Papernumber

Antigen Antibody Visualising Dilution Source Fixative Secondaryantibodyconjugated with

II PGP 9.5 Anti-PGP 9.5 Nerve fibres 1/800 Biogenesis Lana TRITCII Substance P Anti-SP Substance P 1/400 Peninsula Lana TRITCII Calcitonin gene-

related peptideAnti-CGRP CGRP 1/400 Peninsula Lana TRITC

PGP 9.5 = protein gene product 9.5; TRITC = Tetramethyl-rhodamine-isothiocyanate; All antibodies were

polyclonal and 14m thick sections were used.


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27

Six compartments in all specimens were analysed: the epidermis, dermo-epidermal

junction, papillary dermis, reticular dermis, eccrine sweat glands and their ducts and,

wherever applicable, beneath pustules. Each separate fragment of nerve fibre was

considered as one fibre. All five sections were analysed and the mean values per

square millimetre or millimetre of epidermal length were calculated. Image analysis

of the nerve fibres around the sweat glands was performed. The area of the positive

nerves was expressed in per cent of the total sweat gland area.

Three immunofluorescence double stainings were performed (Table 2b).

Table 2b. Double stainings with immunofluorescence techniques.

PGP 9.5 = Protein gene product 9.5; HNL = human neutrophil lipocalinFITC = Fluorescein isothiocyanate; TRITC = Tetramethyl-rhodamine-isothiocyanateSections were 14m thick.*All close contacts between mast cells and nerve fibres in the papillary dermis were counted and thenumber of such contacts per mm of epidermal length was calculated.

Serum immunofluorescence (paper V)

Sections from palmar skin of healthy controls (non-smokers and smokers), 6 m thick

and fixed in acetone, were incubated overnight at +4C with serum (dilution 1/150)

from 45 patients with PPP and 23 patients with palmar eczema. Immunofluorescence

stainings were also performed with sera from 7 patients with myasthenia gravis, all of

whom had elevated serum concentrations of nAChR antibodies. Fluorescein-

isothiocyanate (FITC)-anti-human IgG (dilution 1/40; Dakopatts, Glostrup, Denmark)was used as secondary antibody. Control with FITC anti-human IgG omitting the

patient serum was negative. All parts of the sweat gland apparatus (duct and gland),

epidermis and dermis were studied for the presence of staining. The staining intensity

was classified as weak (+), medium (++) or strong (+++).

Double staining: endothelium palmoplantar pustulosis serum (paper V)

This double staining was performed to confirm that the immunofluorescence obtained

with the PPP sera was localised on endothelial cells. Sections 6 m thick and fixed in

Papernumber

Antibodies against Visualizing Secondary antibodyconjugated with

II Tryptase and PGP 9.5 Contacts mast cells - nerve fibres* Texas Red - FITCII Substance P and HNL Neutrophils containing substance P TRITC - FITCII Substance P and EG2 Eosinophils containing substance P TRITC - FITC


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28

acetone were incubated with two mouse monoclonal anti-human endothelial

antibodies, Q bend 10 (dilution 1/40; Skybio, Bedfordshire, UK) and CD 31 (dilution

1/40; Dakopatts), overnight at 4C. Biotinylated horse antimouse IgG (dilution 1/200;

Vector, CA, USA) was used as secondary antibody. Subsequently the sections were

incubated with Texas Red Streptavidin (dilution 1/100; Vector) for 30 min and then

with 10% normal mouse serum (Dakopatts) for 60 min. The sections were then

allowed to react with 10% normal rabbit serum for 10 min and thereafter with serum

from the patients and FITC-anti-human IgG as above. To rule out non-specific

staining, including overlapping between the fluorescence filters, three control

stainings were performed: one using mouse IgG of the same isotypes and dilutions as

the primary endothelial antibodies plus patient serum, another with mouse IgG and

omitting the patient serum, and as a third control the endothelial antibodies were used

without the patient sera.

Western blot (paper III)

Western blot analysis was run on cell extracts from pure preparations of neutrophils

and eosinophils from peripheral blood from healthy donors. Granulocytes from a

Ficoll preparation were incubated with supermagnetic particles coupled to a

monoclonal antibody against CD 16, a molecule present on neutrophils but not on

eosinophils (Hansel et al 1991). The cell preparations were kindly provided by

Associate Professor Lena Hkansson, Section of Clinical Chemistry, Department of

Medical Sciences, University Hospital, Uppsala. Protein extract from human placenta

was used as a positive control (Rowell and Sastry 1981).

The proteins were separated on 10% SDS-PAGE ready-gels (Bio Rad, CA, USA),

and blotted on a nitrocellulose membrane. ChAT was visualised with a polyclonal

rabbit-anti-ChAT antibody (dilution 1/1,000; Biogenesis, Poole, UK) and an Immun-Blot kit with an alkaline phosphatase conjugate (dilution 1/3,000; Bio Rad). As a

negative control, placenta extract was used as above, but without the primary

antibody.

Radioimmunoassay (paper V)

Serum nAChR antibodies were measured in sera from 45 PPP patients and, for

comparison, in sera from 10 patients with palmar eczema, with a radioimmunoassayused for determination of acetylcholine receptor antibodies in myasthenia gravis


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29

(Lefvert et al 1978). In brief, a preparation of cholinergic receptors from human

skeletal muscle was incubated with radiolabelled alpha-bungarotoxin, serum was

added and the toxin-receptor-IgG complex was precipitated using anti-human IgG.

The precipitate was separated and washed by centrifugation. Radioactivity (CPM)

was determined and the concentration of receptor antibodies in arbitrary units was

calculated.

StatisticsThe statistical significance of differences was calculated by the Mann-WhitneyU -test

(papers I, II, III, IV and V) or Fishers exact test (paper V).


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30

RESULTS AND DISCUSSION

Paper I Anamnestic data

The worsening effect of warm weather and stress in a high proportion of patients

indicated that the sweat gland apparatus might be a possible target for the

inflammation. The fact that 95% of the patients were smokers at the onset of the

disease (at a mean age of 42 years, range 15-66 years) pointed to nicotine as a possible

precipitating factor for the disease (Table 3).

Table 3. Anamnestic data in 59 patients (52 women, 7 men) with palmoplantar pustulosis.

Per centHeredity for -palmoplantar pustulosis 14-psoriasis 22-thyroid disease 22-gluten intolerance 3

Patients with history of -psoriasis 10-thyroid disease 14-gluten intolerance 8-diabetes 7-vitiligo 5-alopecia areata 3

Stress preceding onset of PPP 25Smoker at onset of PPP 95Worsening associated with- hot weather 36- stress 46

Pruritus 95Arthralgia 42

There was a high prevalence of a number of autoimmune diseases among the PPP

patients, as shown in Table 3.

The association between autoimmune thyroid disease and PPP is well known and has

been investigated in detail by Rosn (1988). The majority of the PPP patients with


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31

thyroid disease had hypothyroidism, the prevalence of which in Swedish women is 1.9

percent (Hallengren 1998).

The increased prevalence of coeliac disease in PPP patients has not been reported

previously. The prevalence figures for coeliac disease in the Swedish population have

increased in the last few years since the introduction of screening for antibodies to

gliadin and endomysium and recently also to tissue transglutaminase. Silent coeliac

disease has been diagnosed in 0.3% of Swedish blood donors (Grodzinsky 1996).

Recent data from a screening study of children in Northern Sweden indicate a

prevalence of at least 1% (Carlsson et al 2001). Since January 2001 anamnestic data

have been available for 82 patients with PPP. The prevalence of previously diagnosed

coeliac disease in this extended group is 6%. One of our patients who was found to

have coeliac disease had disabling PPP, but since the introduction of a gluten-free diet,

her PPP has totally cleared, indicating that gluten intolerance might be of pathogenetic

relevance in PPP.

There was also a high prevalence of diabetes among the PPP patients. This has

become even more evident since the number of patients has increased (to 82)

compared with the number at the start of the study. Twelve of the 82 patients (14.6 %)

had diabetes; 9/12 were < 50 years old. At screening for diabetes in the community of

Lax, Sweden, 0.8% of the screened women aged 25-44 years and 2% of those aged

45-54 years were found to have diabetes (type 1 or type 2) (Andersson et al 1991).

Four of the patients (4.8%) had type 1 diabetes. The prevalence of type 1 diabetes

among women in the community of Lax was 0.3-0.4%. Thus there is a marked

increase in the prevalence of diabetes in PPP, which has not been reported previously.

However, a predisposition to diabetes in PPP was discussed in a Japanese study, as a

diabetic pattern at an oral glucose tolerance test was found in 22% of the patients

(Uehara 1983), but there are no other reports on such an association. There are,however, previous reports of an increased prevalence of type 2 diabetes in psoriasis

(Binazzi et al 1975). A significantly raised prevalence of psoriasis and vitiligo has

been reported in children with type 1 diabetes (Montagnani et al 1985).

The high prevalence of associated autoimmune disease in PPP gave us reason to

consider the possibility that PPP itself might be an autoimmune disease affecting the

skin and also the joints.


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32

Clinical findings

Erythema and scaling were present in all but one patient. Fresh pustules were observed

in 26 patients (range 1-100). Patients with the highest cigarette consumption had the

largest mean number of pustules (but there were large variations and the groups of

patients were small).

The association between PPP and autoimmune disease was further strengthened by

the presence of antibodies to thyroglobulin/thyroperoxidase in 25% of the patients.

IgA antibodies to gliadin were present in 25%, compared to 9% in female healthy

blood donors (Lindquist et al, unpublished data). Among patients with psoriasis

vulgaris 16% had IgA antibodies to gliadin (Michalsson et al 1993), thus the

prevalence of IgA antibodies to gliadin is even higher in PPP.

The significantly elevated mean serum IgA and decreased IgM are similar to the

pattern present in coeliac disease and dermatitis herpetiformis (O'Mahony et al 1990)

and also in psoriasis (Michalsson et al 1995) and psoriatic arthritis (Lindqvist el al,

unpublished data), indicating that the intestinal mucosa might also be involved in PPP.

Elevated serum ECP in PPP was previously reported by Lundin et al (1990) and

suggested that the eosinophil granulocyte was activated. The results of the present

study further confirm that eosinophil granulocytes are involved in the inflammation

(see below).

Inflammatory cells

The pustules were found to contain large numbers of eosinophils, an observation not

made previously, as well as neutrophils, indicating that the eosinophils participate in

the pustule formation together with neutrophils. Furthermore, numerous eosinophils

were present in the papillary dermis below the pustules. Another previously

unreported feature was the massive infiltrates of mast cells in the upper dermis,especially in specimens with pustules. One important chemoattractant for both

neutrophils (Baggiolini et al 1989) and activated eosinophils (Burrows et al 1991) is

IL-8, which has been shown to be present in the eccrine duct in general and in the

epidermis in PPP skin (Anttila et al 1992; Jones et al 1995). Furthermore, mast cells

have been found to secrete IL-8 (Ansel et al 1997).

There was a massive infiltrate of lymphocytes in the papillary dermis, with a

tendency to accumulation below the pustule. The accumulation of the participating


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33

inflammatory cells below the pustules may indicate that there is an epidermal target

for the inflammation that is not evenly distributed in the epidermis.

Fig. 3. Schematic drawing of the participating inflammatory cells in PPP.

The sweat gland and duct In the specimens from involved PPP skin no acrosyringia were visible with the

keratin antibody AE1/AE3, reported to give staining in the sweat gland apparatus

(Watanabe et al 1993), in contrast to the findings of acrosyringia in the control

specimens, indicating that the intraepithelial duct may be destroyed in PPP, which

might reflect an inflammatory process at this site. This feature may have pathogenetic

relevance in PPP, since there were no changes in the appearance of the acrosyringia

in specimens from our tylotic eczema patients or in those from patients withdyshidrotic eczema (Kutzner et al 1986). We also stained the sweat pores in the

palmar skin of PPP patients with the iodine-starch method and compared the pattern

with that in the palms of healthy persons (no data shown). In the PPP patients a

diffuse pattern was observed, whereas in the control persons there was a distinct

pattern of black spots in even rows over the entire palm.


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34

Paper II PGP 9.5, substance P and calcitonin gene-related peptide

In view of the seemingly strong influence of stress on the inflammatory activity in

PPP and the itching when new pustules were formed, a study of the distribution of

nerve fibres and the presence of SP- and CGRP-like immunoreactivity (-LI) in

involved skin in PPP patients and in healthy controls was undertaken.

The innervation of the sweat glands was studied with a general nerve marker,

protein gene product 9.5 (PGP 9.5). In the patients the nerve fibres around the sweat

glands seemed to be more or less fragmented, while in the controls they encircled the

sweat gland without any interruptions (Fig. 4a and b). Image analysis showed that

there were significantly fewer fibres around the sweat glands in the patients than in

the controls (p=0.0006).

Fig. 4. PGP 9.5-positive nerve fibres around a sweat gland in involved palmar PPP skin (a) (note thefragmented fibres) and in palmar control skin (b) (x300).

The largest numbers of nerve fibres with SP- and CGRP-LI were observed in the

papillary dermis, both in patients and controls. There was a tendency to a larger number of nerve fibres with SP- and CGRP-LI in the patients, especially in the

papillary dermis. In the reticular dermis nerve fibres with SP- and CGRP-LI were

localised close to blood vessels and some were also observed close to the sweat ducts.

Whether the probable damage to the nerves around the sweat gland is a result of the

inflammation higher up in the papillary dermis is not known. Nor is it known whether

this has an influence on the function of the sweat gland. One possibility is that the

damage may be induced by neurotoxic effects of eosinophil granule proteins, as large


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numbers of eosinophils have been found in the pustules and also in the upper

papillary dermis (Eriksson et al 1998).

Both psoriasis and PPP patients experience worsening of their skin disease during

periods of stress. An increased number of CGRP-positive nerve fibres has been

observed in the papillary dermis of psoriasis patients with high stress levels (Harvima

et al 1993). CGRP induces vasodilation, resulting in long-lasting erythema

(Wallengren and Hkanson 1987). An increased number of SP-positive nerve fibres

(Naukkarinen et al 1989) and an increased number of contacts between SP-containing

nerve fibres and mast cells (Naukkarinen et al 1996) have also been found in psoriatic

lesions, indicating neurogenic involvement in the inflammation in psoriasis.

There are discrepancies in the numbers of nerve fibres reported from different

studies, which may indicate that there are difficulties in counting nerve fibres, and the

results may depend on the methods employed. In this study all nerve fibres were

counted, including nerve fragments, and the number varied considerably, both in

controls and patients. With image analyses, where the number of immunoreactive

nerve fibres could have been related to the area of the epidermis, different results

might have been obtained, since the epidermis in PPP skin is much thicker than in

control palmar skin. There are similar problems in comparing the number of nerve

fibres in the papillary dermis with that in healthy skin.

Nerve fibres and their contacts with mast cells

Close contacts between nerve fibres and mast cells provide an opportunity for

neuropeptides to degranulate these cells, and release of histamine and several other

degranulation products will further enhance the inflammatory reaction. The itching

that occurred when new PPP pustules were formed might be explained by histamine

release caused by neuropeptides.The number of tryptase-positive mast cells in the papillary dermis was larger

(p=0.0003) in the lesional palmar skin from PPP patients than in the healthy controls,

and the number of contacts between mast cells and nerve fibres with PGP 9.5-LI was

also significantly larger (p=0.02).

As the number of nerve fibres was similar in PPP and control skin, but the number

of mast cells was three times larger in the PPP skin than in the controls, the increased

number of contacts may be due to the increase in mast cells.


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It has been reported (Naukkarinen et al 1996) that both the number of mast cells and

the number of SP-positive nerve fibres in contact with mast cells are increased in

psoriatic lesions. The increased number of contacts in PPP and in psoriatic skin may

imply more pronounced neurogenic involvement in the inflammation than in

conditions without a mast cell increase.

Neuropeptide immunoreactivity in granulocytes

PGP 9.5- and SP-LI, but not CGRP-LI, were present in granulocytes. With use of

double staining, neutrophils showed SP-LI, whereas eosinophils were SP-negative.

The neutrophils were situated in the pustule, or were visible in the papillary dermis,

and were observed within the sweat duct in the papillary dermis (possibly migrating

towards the pustule) (Fig. 5).

Fig. 5. SP-positive granulocytes (neutrophils) in the sweat duct, possibly migrating towards the

pustule, and in the pustule (x475).

Substance P has previously been detected in human peripheral leucocytes from

healthy subjects (De Giorgio et al 1998). SP-LI has also been found in neutrophils in

infiltrates in psoriatic lesions (Pincelli et al 1992). This suggests that neutrophils are

another possible source of SP, both in PPP and in psoriatic skin. As there is a massive

granulocyte infiltration in PPP, the possible influence of SP may be more pronounced

in this disease than in psoriasis, although it is probably of importance in both


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conditions. SP has been reported to stimulate proliferation of cultured keratinocytes in

a dose-dependent manner (Tanaka et al 1988). Keratinocytes in PPP skin might be

influenced in a way similar to that in psoriasis, where their proliferation rate is

increased.

Paper IIIChAT and AChE in the epidermis and sweat gland apparatus

The mechanisms underlying the

inflammation in the acrosyringium

in PPP are not known. Sweating

and the sweat gland apparatus

seem to play an important role in

the pathogenesis of PPP.

Sympathetic fibres innervate

the sweat glands, but are cholinergic,

and ACh is the main inducer of

sweating (Fig. 6). Fig. 6.

As the ACh-synthesising enzyme,

choline acetyltransferase, and the degrading enzyme, acetylcholinesterase, regulate

the ACh level, the distribution of ChAT- and AChE-LI was studied in normal palmar

skin in non-smokers and smokers and in involved skin in PPP patients.

In addition to observing ChAT- and AChE -LI in the epidermis, we found that the

eccrine glands and ducts displayed more intense ChAT and AChE reactivity than the

epidermis. Table 4 shows some important findings of the ChAT and AChE stainings.


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Table 4. Results of the ChAT and AChE immunohistochemistry in the epidermis andsweat gland apparatus.

ChAT non-smokers ChAT smokers ChAT involved

PPP

AChE non-smokers,

smokers and involved

PPPStaining intensity/vital epidermis Weak to moderate Weak to moderate Weak to

moderate

Weak

Staining intensity/acrosyringium

in stratum corneum

No staining No staining No staining Strong staining

Number of positive acrosyringia

in vital epidermis and the

presence of strongly stained

acrosyringia

Larger number than in

the smokers and PPP

patients and some were

strongly stained

Larger number than in

the PPP patients and

none were strongly

stained

Smallest number

and none were

strongly stained

No differences between

the groups, almost half of

them were strongly

stained.

Number of strongly stained coils

and reticular ducts

Largest number Medium number Smallest number No differences between

the groups

There were some differences between the distribution of ChAT- and that of AChE-LI.

In the acrosyringium AChE-LI was most intense in the lower part of the stratum

corneum corresponding to the site of the pustule in PPP. This is of special interest,

since there is a homology between AChE and thyroglobulin (Malthiery and Lissitzky

1987), against which many patients have antibodies. The carboxy terminal of thyroglobulin shows up to 64% homology with AChE. (The sites of thyroid hormone

synthesis are clustered at both ends of the thyroglobulin.) No ChAT-LI was present in

the acrosyringium at this level. On the other hand there was marked ChAT-LI in the

acrosyringium in the living part of the epidermis. Klapproth et al (1997) reported that

ChAT enzyme activity and ChAT-LI were to some extent correlated and that high

ChAT enzyme activity and immunoreactivity corresponded to a high ACh content

(skin, intestine) and vice versa (bronchi). Grando et al (1993b) observed ChAT-LIthroughout the epidermis and in epidermal appendages and also found that

keratinocytes have ChAT activity resulting in ACh production. If this is also true for

eccrine sweat glands and ducts, ACh will be produced in both the coil and duct until

it reaches the horny layer.

In the epidermis Grando et al (1993b) found AChE reactivity mainly in the basal

layer (keratinocytes and melanocytes). This was also observed in our specimens,

although the AChE reactivity was much stronger in the eccrine sweat apparatus than

in the interappendageal epidermis.


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ChAT and AChE in inflammatory cells

Granulocytes, which are abundant in the pustules and the papillary dermis in involved

PPP skin, showed strong ChAT-LI. Western blot analysis of proteins extracted from

purified non-stimulated neutrophils and eosinophils confirmed the presence of ChAT-

like proteins in large amounts in neutrophils and in very small amounts in

eosinophils. Placental extract was used as a positive control (Fig. 7).

Fig. 7. Western blot with a polyclonal ChAT antibody; Pla=positive control, placenta (5 g protein), Neu=neutrophils (7 g protein), Eos=eosinophils (8 g protein). Single arrow indicates ChAT 54 kDand double arrows ChAT 69 kD (dimer).

In the skin there are normally few, if any, neutrophils and eosinophils. Thus noconclusions can be drawn from our results as to the extent to which the ChAT

reactivity in granulocytes in the PPP patients might have been influenced by nicotine.

If there is ChAT activity in these cells, this might be of importance not only regarding

the inflammation in PPP but also in other inflammatory diseases, e.g. psoriasis, atopic

dermatitis and asthma, where these cells play a significant role. Further studies will

be undertaken to see whether ChAT in granulocytes can synthesise ACh.

In the healthy non-smokers and in the PPP patients AChE-LI was seen in 25% of the mast cells, while only 10% of the mast cells in the smoking controls showed

AChE reactivity. This preliminary finding might indicate that smoking can influence

the AChE activity. There are no reports on AChE+ mast cells in the skin and only one

report on the presence of AChE in human mast cells, namely in a colonic biopsy

specimen where AChE mRNA expression was detected in FcRI-positive cells

(Nechushtan et al 1996). There are several reports, however, on interactions between

ACh, histamine and mast cells in inflammatory reactions [reviewed in Masini et al

(1985)].


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Paper IV

Nicotinic receptors in the epidermis and sweat gland apparatus

A role of nicotine in the pathogenesis of PPP has long been discussed. Several

diseases are thought to be caused or aggravated by smoking, but the mechanisms

underlying this effect are not known. That nicotine plays a role in this respect has

been reported both in psoriasis vulgaris (Plunkett and Marks 1998) and in psoriatic

arthritis (Averns et al 1996). The skin disease with the most obvious association with

smoking is palmoplantar pustulosis. We showed in studies I and II that the target for

the inflammation in PPP is the acrosyringium. Nicotine acts as an agonist on nicotinic

acetylcholine receptors and can influence a variety of cellular functions, like cell

adhesion and motility (Grando et al 1995a) and keratinocyte differentiation (Grando

et al 1996).

In this study we chose to investigate the expression of the two nAChR subunits-3

and -7, as most of the possible variations of nAChRs on keratinocytes would then be

included.

In healthy subjects the epidermis and, to an even greater extent, the eccrine sweat

gland and its duct expressed both the-3 and -7 subunits of the nAChR. It was also

evident that smoking influenced the staining intensity but not the distribution in

the healthy controls.

The strongest epidermal -3 staining was present in the involved PPP skin, where it

was significantly more intense than in the non-smoking controls (Fig. 8a and b).

a. b.

Fig. 8. Schematic drawings of -3 staining in palmar skin from (a) a non-smoking control: 1= weak staining of the acrosyringium, 2= epidermis with weak, even staining, 3= the entrance of the sweatduct in the epidermis results in a thicker epidermis, 4= weak staining of endothelial cells; and (b)involved PPP skin: 1= epidermis with the strongest staining in the upper part of the stratum spinosum,2= the pustule with stained granulocytes, 3=-3-positive granulocytes in the papillary dermis.


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The strongest staining in the sweat gland apparatus was also seen in the involved

skin. The highest proportion of strongly stained ducts in the reticular dermis in

relation to the area of the sections was again observed in involved PPP skin and this

proportion was significantly larger than in the smoking controls (p=0.01), where the

staining was weakest. Psoriasis specimens were also stained for comparison, and a

pattern similar to that in the PPP specimens was observed (data not shown).

The strongest staining of the -7 subunit in the healthy controls (smokers and non-

smokers) was noted in the keratinocytes in the stratum granulosum, with the most

pronounced intensity in the acrosyringium (Fig. 9a).

In the involved PPP skin, in which the stratum granulosum was abolished, there was

a remarkably different pattern, especially around the acrosyringium and closest to the

pustule, where the surface of the keratinocytes was strongly stained, with a fishnet-

like appearance (Fig. 9b).

a. b.

Fig. 9. Schematic drawings of -7 staining in palmar skin from (a) a non-smoking control: 1= thestrongest staining is seen in the acrosyringium in the stratum granulosum, 2= the strong staining of thestratum granulosum, 3= the entrance of the sweat duct in the epidermis results in a thicker epidermis,4= staining of endothelial cells in the dermis; and (b) involved PPP skin: 1= the fish-net like staining pattern in the epidermis, 2= unstained cells in the pustule, 3= the strong staining of endothelial cells,4= the strongest staining of epidermis is seen closest to the pustule.

The abnormal distribution of the -7 nAChR in the keratinocytes in PPP skin may

affect the differentiation, since keratinocytes are able to synthesise, release and

degrade ACh (Grando et al 1993b). Nicotinic receptors have been found to be

important for the terminal differentiation of keratinocytes. Long-term exposure of

keratinocytes to nicotinein vitrohas been shown to inhibit their migration and to

increase the number of keratinocytes forming cornifying envelopes and also the

expression of filaggrin, involucrin and transglutaminase type 1 (Grando et al 1996).


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Paper V

Serum antibodies to nicotinic receptors and the immunofluorescence pattern

With regard to the massive inflammatory reaction in PPP skin and the high

prevalence of autoimmune disease, it might be suspected that PPP could be an

expression of one or several autoimmune reactions induced by smoking.

A first step in the testing of this hypothesis was to determine whether antibodies

against nAChR could be detected in PPP serum with the method usually used in

Sweden when diagnosing myasthenia gravis (Lefvert et al 1978).

Increased concentrations of such antibodies, though less increased than in

myasthenia gravis, was found in 19/45 (42%) of the PPP patients. The mean antibody

level in the positive sera was 0.75 arbitrary units/l (range 0.2-3.1). Values below 0.2

were considered normal. Among a Swedish group of patients with myasthenia gravis,

93 % had nAChR ab, with a mean value of 3.60+2.20 (Lefvert et al 1978). None of

the sera from the patients with eczema (non-smokers or smokers) had any nAChR ab,

which rules out the possibility of a non-specific reaction in PPP associated with an

inflammation in palmar skin.

Fig. 10. The antibodies (ab) in PPP sera were mostly found in the patients without thyroid and/or gliadin antibodies, indicating that there are two subgroups of PPP.

Twenty-one of the 45 sera (46.7 %) produced a special fluorescence (IF) pattern on

some cells in the papillary dermis in normal palmar skin from a non-smoker. The IF

staining often formed a chain-like pattern (see cover page).

Of the sera from PPP patients with nAChR antibodies 68% gave this positive IF. On

double staining with a specific anti-human endothelial antibody, the IF was found to

be localised to endothelial cells. Furthermore, in palmar skin from a smoker positive

IF was also observed in the acrosyringium in the upper part of the living epidermis.

nAChR ab no nAChR ab0

20

40

60

80

100

thyroid abthyroid and gliadin ab

gliadin ab

no thyroid or gliadin ab

n=19 n=26

P e r c e n

t


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Of the sera from PPP patients without nAChR, the same IF pattern was produced by

31%, although mostly with lower intensity (Fig. 11a and b). Sera that had antibodies

for both nAChR and/or thyroperoxidase/thyroglobulin/gliadin gave the strongest IF.

a. b.Fig. 11. (a) More sera from patients with nAChR antibodies (ab) than from patients without suchantibodies gave immunofluorescence staining. (b) The sera with nAChR antibodies produced stainingof stronger intensity than those without such antibodies.

The strong IF staining in the acrosyringium in palmar skin from a smoker illustrates

the important role of nicotine, since the acrosyringium is the main target of the

inflammation. This strengthens our theory of a possible up-regulation of an antigen

by smoking. The strong IF staining with sera from patients with several types of autoantibodies might be due to an overlap between these different antigens. This

might explain the association between smoking and PPP and possibly also between

PPP and autoimmune thyroid disease and coeliac disease.

Only two (8.7 %) of the 23 sera from patients with palmar eczema produced any

positive structures one with weak (non-smoking man) and one with medium

(previously smoking woman) staining intensity - in the papillary dermis.

Sera from 7 patients with myasthenia gravis with high concentrations of nAChR antibodies did not produce any positive IF, indicating that the autoantigen(s) in PPP

and myasthenia gravis are probably not the same.

The results of this study indicate that PPP is an autoimmune disease. The antibodies

that bind to endothelial cells and to the sweat gland duct may play a pathogenetic role

in PPP by activating endothelial cells and enhancing the massive infiltration of

inflammatory cells in the papillary dermis and the migration of granulocytes outwards

into the acrosyringium in the stratum corneum and into the pustules.

nAChR ab no nAChR ab0

20

40

60

80

100stainedunstained

n=19 n=26

P e r c e n

t

nAChR ab no nAChR ab

0

1

2

3

n=19 n=26

S t a i n i n g

i n t e n s

i t y


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CONCLUSIONS

* The massive infiltrates of lymphocytes

patogenesis ppp

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